Project Summary. Infectious diarrhea constitutes a major endemic health threat as an increasingly frequent and deadly source of food- and water-borne illness. Enterohemorrhagic Escherichia coli (EHEC) are especially significant bacterial pathogens because their toxins can cause a type of renal failure (hemolytic uremic syndrome) for which therapy is limited. The molecular mechanisms by which EHEC and the closely related enteropathogenic E. coli (EPEC), as well as Salmonella enterica serovar Typhimurium, a common cause of human gastroenteritis, inhibit host innate immune responses to promote bacterial colonization are under intense investigation. Understanding these host-pathogen dynamics in molecular detail may enhance the development of pharmacological approaches to prevent and treat infections. Through studies of the mechanism of the NleB virulence protein expressed by E. coli and by a related pathogen of mice, Citrobacter rodentium, it was determined that NleB functions as a glycosyltransferase enzyme that covalently modifies host proteins with N-acetylglucosamine (GlcNAc) to subvert their normal functions. NleB disrupts tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) signaling, leading to inhibition of the pro-inflammatory NF-?B pathway. The glycolysis enzyme GAPDH functions as a co- activator of TRAF2 activity. The modification of GAPDH with GlcNAc by NleB prevents GAPDH from binding to and activating TRAF2, leading to a reduced NF-?B response to infection and thus enhanced bacterial survival. It was also discovered that some NleB orthologs modify the arginine residues of host proteins, rather than the expected modification of their serine/threonine residues. S. Typhimurium strains also encode from 1-3 NleB orthologs named SseK/1/2/3, although their enzymatic activities and relative contributions to virulence are less clear. The proposed project is designed to elucidate the molecular mechanisms of NleB/SseK glycosyltransferase activities, with the ultimate goal of using these data to inform future strategies to prevent and treat infectious diarrhea. The specific aims are: 1) Elucidate the NleB/SseK GlcNAcylation mechanisms and their targeting of host proteins. 2) Identify GAPDH GlcNAcylation sites and characterize their role in GAPDH activity and NF-?B pathway activation. This multidisciplinary approach provides an opportunity to probe not only the enzymology of these virulence factors, but also to effect the future development of small molecules with anti-virulence properties.